1. Field of the Invention
The invention relates to the field of antibodies, and particularly to modified antibodies, methods of preparing modified antibodies and uses thereof. More particularly, the invention relates to the preparation of more active IgG antibodies by the addition of an extra immunoglobulin domain to the constant region.
2. Background and Related Art
For several decades antibodies have been indispensable in research and diagnosis and more recently in the therapeutic treatment of diseases due to their specific binding properties and high stability. Monoclonal antibodies were initially produced by fusing a chosen B cell line with an immortal myeloma cell line to produce hybridomas, immortal cells that secrete only the selected antibody type of the selected B cell clone. The use of recombinant DNA technologies has enabled new methods of producing antibodies as well as the design of new antibody constructs.
Structurally, each antibody is formed by the interaction of two identical “heavy” chains and two identical “light” chains, all of which combine to form a Y shape molecule (the heavy chains span the entire Y, and the light chains the two arms only). An immunoglobulin G antibody molecule contains complementary determining regions (CDRs) which determine antigen binding, constant regions that determine effector function and framework regions. An antibody construct can include any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule, such as but not limited to at least one CDR of a heavy or light chain or a ligand binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework region, or any portion thereof. An antibody fragment can include the fragment of the immunoglobulin molecule known as the Fab containing the CDR antigen binding site, generated by cleavage of the antibody with the protease papain which cuts at the “hinge” region of the Y shaped antibody molecule producing two Fab fragments. An antibody can include or be derived from any mammal, such as but not limited to a human, a mouse, a rabbit, a rat, a rodent, a primate, or any combination thereof.
Antibodies (Abs) to human antigens usually do not cross-react with the corresponding rodent antigen, with the exception being some Abs to antigens that are highly conserved in structure. Consequently, while developing an Ab to a human target, there is often a need for a separate Ab to the rodent antigen for the purpose of performing preclinical studies in rodents. Because such studies are done to reveal what could be expected to happen in humans treated with the anti-human Ab, it is important that the anti-rodent “surrogate” Ab being used in the animal studies is similar to the anti-human Ab in as many characteristics as possible. Such characteristics for an Ab may include affinity or avidity for antigen, relative neutralization potency, isotype and the associated Fc-mediated immune effector functions (e.g. complement fixation), pharmacokinetic behavior, and ability to form immune complexes with its soluble target antigen. Because there are usually few choices of Abs that can serve as a suitable surrogate for animal studies, it is often very difficult, if not impossible, to find the perfect surrogate Ab. It may be that the two most important characteristics, neutralization of rodent antigen bioactivity and analogous IgG isotypes, are considered sufficient for a surrogate Ab.
While it is possible to develop a rodent antibody that neutralizes the corresponding rodent antigen, it is often necessary to change the antibody isotype to satisfy one criterion for it serving as a surrogate Ab for the human antibody: having its isotype be the functional counterpart isotype to the human antibody. In doing so, it has been shown that the resulting modified antibody may demonstrate in vivo bioactivity in vitro and show complement-fixing activity against rodent antigen-expressing cells in vitro. However, the amount of the modified antibody required to block a given amount of antigen bioactivity can be much higher than than the amount of human antibody required to block the same amount of human antigen bioactivity. Furthermore, the modified rodent antibody can be less potent than the original rodent antibody against the rodent antigen.
This difference in activity and potency can be the result of a fundamental difference between the way the modified antibody and the human antibody bind antigen. Whereas both arms of the human antibody can simultaneously bind two different antigen molecules, the binding of one arm of the dimeric modified antibody molecule to one antigen molecule can prevent the second arm from binding to a second antigen molecule. The modified antibody may be functionally monovalent whereas the natural antibody may be bivalent. Further, by virtue of its ability to bind two molecules of a target that itself may be a homopolymer (for example, TNF is a homotrimer) that can be bound by more than one molecule, the natural antibody may be capable of forming higher order complexes with the target molecule. In contrast, because of its inability to bind more than one target molecule simultaneously, the modified antibody would not be capable of forming higher order complexes with the target molecule. The relative stability of the natural antibody/target molecule complexes and the modified antibody/target molecule complexes would therefore be expected to be dramatically different, since most molecules of the natural antibody would be bound to the complex bivalently and have a very slow dissociation rate, whereas each molecule of the modified antibody would be bound monovalently and therefore have a much faster dissociation rate. Because dissociation of the modified antibody from the target results in a target molecule that is free and bioactive, the result would be large differences in neutralization potencies between natural and modified antibodies.
In addition to neutralization potencies, the difference in the size and complexity of the Ab/target molecule complexes would also be expected to affect such activities as serum clearance rates and Fc receptor binding affinities with concomitant cell activation.
Thus, in engineering modified antibodies it is sometimes desirable to ensure that the resulting construct is functionally bivalent by virtue of its ability to bind two molecules of a target. In the case of an antigen that is itself a homopolymer that can be bound by more than one antibody molecule, it is desirable to have a construct that is capable of forming higher order complexes with the antigen in order to achieve maximum potency and stability of the antibody/antigen complex.
Thus, there is a need for a method of engineering antibodies to provide added flexibilty and spatial distance to allow for multiple binding valencies and complex formations in antigen/antibody binding resulting in both favorable binding characteristics and neutralization capabilities of the antibody construct.